Successful Hematopoietic Stem Cell Transplantation in 2 Children with X-Linked Chronic Granulomatous Disease from Their Unaffected HLA-Identical Siblings Selected Using Preimplantation Genetic Diagnosis Combined with HLA Typing

Abstract

We report 2 children with X-linked chronic granulomatous disease (X-CGD) who underwent hematopoietic stem cell transplantation (HSCT) using grafts from their siblings selected before implantation to be both unaffected and HLA-matched donors. Preimplantation genetic diagnosis (PGD) along with HLA-typing were performed on preimplantation embryos by single-cell multiplex polymerase chain reaction using informative short tandem repeat markers in the HLA locus together with the gene region containing the mutations. Two singleton pregnancies resulted from the intrauterine transfer of selected embryos; these developed to term, producing 1 healthy female and 1 X-CGD carrier female, which are HLA-identical siblings to the 2 affected children. Combined grafts of umbilical cord blood (UCB) and bone marrow (BM) stem cells were administered to the recipients after myeloablative (MA) conditioning at the ages of 4.5 years and 4 years, respectively. Both patients are well, with complete donor hematopoietic and immunologic reconstitution, at 18 and 13 months posttransplantation, respectively. This report demonstrates that HSCT with HLA-matched sibling donors created by PGD/HLA typing of in vitro fertilized embryos is a realistic therapeutic option and should be presented as such to families with children who require a non-urgent HSCT but lack an HLA-genoidentical donor. We report 2 children with X-linked chronic granulomatous disease (X-CGD) who underwent hematopoietic stem cell transplantation (HSCT) using grafts from their siblings selected before implantation to be both unaffected and HLA-matched donors. Preimplantation genetic diagnosis (PGD) along with HLA-typing were performed on preimplantation embryos by single-cell multiplex polymerase chain reaction using informative short tandem repeat markers in the HLA locus together with the gene region containing the mutations. Two singleton pregnancies resulted from the intrauterine transfer of selected embryos; these developed to term, producing 1 healthy female and 1 X-CGD carrier female, which are HLA-identical siblings to the 2 affected children. Combined grafts of umbilical cord blood (UCB) and bone marrow (BM) stem cells were administered to the recipients after myeloablative (MA) conditioning at the ages of 4.5 years and 4 years, respectively. Both patients are well, with complete donor hematopoietic and immunologic reconstitution, at 18 and 13 months posttransplantation, respectively. This report demonstrates that HSCT with HLA-matched sibling donors created by PGD/HLA typing of in vitro fertilized embryos is a realistic therapeutic option and should be presented as such to families with children who require a non-urgent HSCT but lack an HLA-genoidentical donor. IntroductionX-linked chronic granulomatous disease (X-CGD) is an inherited defect in phagocyte nicotinamide adenine dinucleotide phosphate oxidase resulting from a deficiency of gp91phox component. At the molecular level, X-CGD is caused by mutations affecting the CYBB gene on chromosome Xp21.1 [1Heyworth P.G. Cross A.R. Curnutte J.T. Chronic granulomatous disease.Curr Opin Immunol. 2003; 15: 578-584Crossref PubMed Scopus (332) Google Scholar, 2Segal B.H. Leto T.L. Gallin J.I. et al.Genetic, biochemical, and clinical features of chronic granulomatous disease.Medicine (Baltimore). 2000; 79: 170-200Crossref PubMed Scopus (720) Google Scholar]. As a result of impaired phagocytic ability, affected persons suffer from severe recurrent infections, both bacterial and fungal, resulting in the formation of infiltrating granulomatous lesions. Despite prophylactic therapy, long-term survival remains poor; only 50% of patients are alive at the age of 30 years [3Van den Berg J.M. van Koppen E. Ǻhlin A. et al.Chronic granulomatous disease: The European experience.PLoS ONE. 2009; 4 (e5234:1–10)Crossref Scopus (497) Google Scholar, 4Winkelstein J.A. Marino M.C. Johnston Jr., R.B. et al.Chronic granulomatous disease. Report on a national registry of 368 patients.Medicine (Baltimore). 2000; 79: 155-169Crossref PubMed Scopus (1223) Google Scholar]. Allogeneic hematopoietic stem cell transplantation (HSCT) using an HLA-matched sibling donor (MSD) has achieved long-term cure of X-CGD in 80% to 90% of cases [5Seger R.A. Güngor T. Belohradsky B.H. et al.Treatment of chronic granulomatous disease with myeloablative conditioning and an unmodified hemopoietic allograft: a survey of the European experience, 1985-2000.Blood. 2002; 100: 4344-4350Crossref PubMed Scopus (226) Google Scholar, 6Seger R.A. Modern management of chronic granulomatous disease.Br J Haematol. 2008; 140: 255-266Crossref PubMed Scopus (194) Google Scholar]. A healthy MSD is not available in the vast majority of the cases, however. For such patients, a matched unrelated donor (MUD) is a suitable option, but at the cost of increased teatment-related morbidity and mortality (TRM) [5Seger R.A. Güngor T. Belohradsky B.H. et al.Treatment of chronic granulomatous disease with myeloablative conditioning and an unmodified hemopoietic allograft: a survey of the European experience, 1985-2000.Blood. 2002; 100: 4344-4350Crossref PubMed Scopus (226) Google Scholar, 7Del Giudice I. Iori A.P. Mengarelli A. et al.Allogeneic stem cell transplant from HLA-identical sibling for chronic granulomatous disease and review of the literature.Ann Hematol. 2003; 82: 189-192PubMed Google Scholar]. Preimplantation genetic diagnosis (PGD) for single-gene diseases was developed to help families desiring a new pregnancy to select unaffected embryos in vitro, thereby avoiding the need for pregnancy termination. Recently, this strategy has been combined with HLA typing with the aim of selecting suitable histocompatible embryos as potential stem cell donors for affected siblings [8Fiorentino F. Biricik A. Karadayi H. et al.Development and clinical application of a strategy for preimplantation genetic diagnosis of single gene disorders combined with HLA matching.Mol Hum Reprod. 2004; 10: 445-460Crossref PubMed Scopus (70) Google Scholar, 9Van de Velde H. Georgiou I. De Rycke M. et al.Novel universal approach for preimplantation genetic diagnosis of β-thalassemia in combination with HLA matching of embryos.Hum Reprod. 2004; 19: 700-708Crossref PubMed Scopus (92) Google Scholar, 10Kuliev A. Rechitsky S. Tur-Kaspa I. et al.Preimplantation genetics. Improving access to stem cell therapy.Ann N Y Acad Sci. 2005; 1054: 223-227Crossref PubMed Scopus (47) Google Scholar]. There is limited experience with this methodology and even less with its completed clinical application, however.MethodsPatient CharacteristicsPatient 1In this 4.5-year-old boy with an established diagnosis of X-CGD since infancy, diagnosis was confirmed by respiratory burst screening tests and by DNA CYBB gene sequencing (R73X C → T mutation). Despite both antimicrobial and interferon-γ prophylaxis, the patient had recurrent infections from the age of 4 months onward, and granulomatous colitis was diagnosed at the age of 1.7 years.Patient 2This 4-year-old boy was diagnosed with X-CGD at age 6 months by both functional and molecular tests (R157X C → T mutation). Before presenting for HSCT, he had experienced multiple cervical abscesses despite receiving antibiotic prophylaxis.Donor SelectionNeither patient had an HLA-genoidentical sibling donor. The patients' parents came to the Diagnostic Genetic Centre in Athens on their own initiative for genetic consultation. After detailed genetic counseling, the couples came to the Centre for Human Reproduction, Genesis Athens Hospital for complete counseling regarding in vitro fertilization (IVF) and embryo biopsy procedures. The clinical protocol was approved by the center's Institutional Research and Ethics Committee for the IVF treatment, embryo biopsy, and PGD procedures required for the transfer of unaffected and HLA-identical embryos to the mother's uterus. Written informed consent concerning the PGD/HLA typing procedure, as well as the cryopreservation of nontransferred embryos for future use, in accordance with the Helsinki Declaration, was obtained from both couples. A single cycle of IVF was initiated for each couple, and standard hormonal stimulation and culture techniques were used to generate embryos for PGD and HLA genotyping [11Pantos K. Meimeth-Damianaki T. Vaxevanoglou T. et al.Prospective study of a modified gonadotropin-releasing hormone agonist long protocol in an in vitro fertilization program.Fertil Steril. 1994; 61: 709-713PubMed Scopus (37) Google Scholar, 12Palermo G. Joris H. Devroey P. et al.Pregnancies after intracytoplamic injection of single spermatozoon into an oocyte.Lancet. 1992; 340: 17-18Abstract PubMed Scopus (2874) Google Scholar]. All fertilized embryos were cultured and biopsied by laser on day 3 in the first couple and on day 5 for the second couple [13Kokkali G. Traeger-Synodinos J. Vrettou C. et al.Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation diagnosis of β-thalassemia: a pilot study.Hum Reprod. 2007; 22: 1443-1449Crossref PubMed Scopus (118) Google Scholar]. Embryo genotyping was performed using a nested multiplex polymerase chain reaction (PCR) approach, involving first- and second-round amplification of the informative polymorphic short tandem repeat (STR) markers previously selected by extensive HLA genotyping and haplotype analysis of the family members. Detection of the disease mutation was performed by minisequencing [14Pangalos C.G. Hagnefelt B. Kokkali G. et al.Birth of a healthy histocompatible sibling following preimplantation genetic diagnosis for chronic granulomatous disease at the blastocyst stage coupled to HLA typing.Fetal Diagn Ther. 2008; 24: 334-339Crossref PubMed Scopus (11) Google Scholar]. Embryos identified to be HLA-identical to the patients and unaffected by X-CGD (homozygous wild-type allele or heterozygous mutation) were transferred 6 days after fertilization to the uterus of the respective mothers. The 2 singleton full-term pregnancies resulted in the birth of 1 healthy and 1 X-CGD carrier female siblings. Confirmatory genetic testing was performed using chorionic villus sampling in the first trimester for CGD mutations and HLA typing. To rule out the possibility of extreme inactivation of the X chromosome in the case of the heterozygotic sibling donor carrying the nonmutated CYBB, postnatal neutrophil superoxide production was measured.HSCTUmbilical cord blood (UCB) was collected, frozen, and stored in liquid nitrogen as described previously [15Rubinstein P. Dobrila L. Rosenfield R.E. et al.Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution.Proc Natl Acad Sci USA. 1995; 92: 10119-10122Crossref PubMed Scopus (706) Google Scholar]. Given the limited experience with UCB transplantation (UCBT) in patients with X-CGD, we decided to combine UCB and bone marrow (BM) HSC from the same donors, to ensure stable long-term engraftment. Therefore, after parental written consent was provided, when the 2 sibling donors reached age 16 months, reduced-volume BM was harvested. Both patients were conditioned with busulfan (Bu; 16 mg/kg i.v) and cyclophosphamide (Cy; 200 mg/kg i.v.). Graft-versus-host disease (GVHD) prophylaxis comprised cyclosporine A and a short course of methotrexate (MTX). Time to white blood cell engraftment was defined as the first of 3 consecutive days with a neutrophil count > 0.5 × 109/L, and platelet engraftment was defined as the first of 3 consecutive days on which a platelet count > 20 × 109/L was maintained without transfusion. To assess engraftment and the degree of chimersm, patients were monitored at different time points, using a PCR assay to identify genetic polymorphism of STRs. Superoxide production by neutrophils was measured by the dihydrorhodamine (DHR) test. Expression of gp91phox on neutrophils was measured using the 7D5 monoclonal antibody (mAb) and flow cytometry.ResultsPGD/HLA Typing of EmbryosEach family was subjected to a single clinical IVF cycle. A total of 48 embryos were tested for the 2 couples. Eight of these embryos were diagnosed as unaffected and HLA-matched, and 2 embryos from each cycle were transferred, resulting in 2 singleton pregnancies and 2 newborns, 1 healthy and 1 X-CGD carrier. All X-CGD–unaffected embryos not transferred were cryopreserved for eventual future use. Table 1 summarizes the PGD/HLA typing results.Table 1Summary of the PGD/HLA typing resultsFamilyIVF CycleEmbryos FormedHLA-Matched, CGD-Unaffected EmbryosEmbryos TransferredPregnancies11376∗Four embryos were homozygous for the wild-type allele.2121112†Both embryos were carriers for the CGD mutation.21PGD indicates preimplantation genetic disease; CGD, chronic granulomatous disease. Biopsies were performed at the cleavage stage (day 3 after fertilization) for family 1 and at the blastocyst stage (day 5 after fertilization) for family 2.∗ Four embryos were homozygous for the wild-type allele.† Both embryos were carriers for the CGD mutation. Open table in a new tab Graft CharacteristicsThe numbers of UCB-nucleated and CD34+ cells collected from the first donor (1.4 × 107/kg and 3.5 × 104/kg, respectively) were deemed insufficient to secure long-term engraftment. Therefore, we decided to increase the number of hematopoietic progenitors by harvesting a reduced BM volume from the same donor. In the case of second donor, and despite an adequate number of cells in the UCB unit, we decided to harvest a small volume (100 mL) of BM to further enrich the number of hematopoietic progenitors. Coinfusion of BM cells in the second case was of the limited experience with matched in X-CGD and the of in small of children unrelated P. et of chronic granulomatous disease after second unrelated donor umbilical cord blood 2007; PubMed Scopus Google Scholar]. In both cases, BM was when the donors reached the age of 16 on the number of and cells of the grafts are in Table of patients, donors, and and transplantation carrier volume collected, × × CD34+ × × volume collected, × × CD34+ × × indicates chronic granulomatous disease; umbilical cord bone disease. Open table in a new tab patients after receiving a combined of UCB and BM engraftment was achieved on day in patient 1 and on day in patient and platelet engraftment on days and respectively. hematopoietic was after day by STRs. patient developed or chronic disease and were at 6 months 1 had since day posttransplantation, of In patient both and gp91phox were to on of the neutrophils posttransplantation, in patient only 50% of neutrophils and gp91phox testing at time At their both patients were in clinical hematopoietic and immunologic at 18 and 13 months posttransplantation, of an with the of using as an HSC donor for an affected sibling has been for years for the of bone and in Scholar]. In the years, parents to by and for an et S. et al.Preimplantation diagnosis for combined with PubMed Scopus Google HLA typing combined with PGD in a case of only embryos for X-linked be both and the of a matched donor using PGD/HLA typing is that with a a limited number of PGD HLA typing on preimplantation embryos experience with the combination of IVF and PGD to pregnancy and birth of a healthy from different children after PGD/HLA the pregnancy embryo transfer was on on the IVF and age of the A. Rechitsky S. Tur-Kaspa I. et al.Preimplantation genetics. Improving access to stem cell therapy.Ann N Y Acad Sci. 2005; 1054: 223-227Crossref PubMed Scopus (47) Google Scholar, de Velde H. De Rycke M. De C. et experience of European preimplantation genetic diagnosis on Reprod. 2009; 24: PubMed Scopus (47) Google Scholar, I. et the of genetic diagnosis with in 2009; PubMed Scopus Google Scholar]. All children were healthy and HLA-identical to their affected siblings. In these only to the cure of affected siblings been de Velde H. De Rycke M. De C. et experience of European preimplantation genetic diagnosis on Reprod. 2009; 24: PubMed Scopus (47) Google Scholar, et hematopoietic stem cell transplantation for from unaffected sibling selected using preimplantation genetic 2004; PubMed Scopus Google Scholar, B. et umbilical cord blood transplantation for using genetic diagnosis for HLA-matched J Hematol. 2004; PubMed Scopus Google Scholar]. on HLA typing been the possibility of of HLA typing or PGD resulting from and be de Velde H. De Rycke M. De C. et experience of European preimplantation genetic diagnosis on Reprod. 2009; 24: PubMed Scopus (47) Google Scholar, L. A. Traeger-Synodinos J. et of and in Reprod. 2009; 24: PubMed Scopus Google Scholar]. Therefore, be to the family that diagnosis is an to rule out an affected In the of this a number of and such as the of for use, of which be by genetic and counseling of the parents G. I. on preimplantation genetic diagnosis for HLA typing to a future as a donor of stem cells to a Reprod. 2002; PubMed Scopus Google Scholar, for 2004; PubMed Scopus Google Scholar, I. de Velde H. The future of preimplantation genetic 2007; PubMed Scopus Google Scholar]. the of healthy embryos that the number created to The on should regarding the of PGD/HLA typing and the of healthy In the transfer of selected embryos on HLA typing in combination with in this is not further is required to the and that be to the application of this of HSC modified to gp91phox has been with in a patients, the and of gene in X-CGD of et production of after gene of chronic granulomatous Natl Acad Sci USA. PubMed Scopus Google Scholar, M. K. et of X-linked chronic granulomatous disease by gene therapy, by of or PubMed Scopus Google Scholar]. HSCT from an HLA-matched donor is the only therapeutic with potential for patients with Allogeneic HSCT is performed at an age in children with recurrent infections despite antimicrobial prophylaxis R.A. Modern management of chronic granulomatous disease.Br J Haematol. 2008; 140: 255-266Crossref PubMed Scopus (194) Google Scholar, in for chronic granulomatous 2009; PubMed Scopus Google Scholar]. A study of the of HSCT in 20 patients with X-CGD that the of donor or had on transplantation E. et donor and HLA-identical sibling stem cell transplantation cure chronic granulomatous disease with long-term and J Haematol. 2009; PubMed Scopus Google Scholar]. is that the and of should be in of a is available for only to of the patients in need of a donor, even in patients of F. for hematopoietic stem cell donors for 2008; PubMed Scopus Google Scholar]. with or unrelated UCB as the of HSC for patients with X-CGD is and only a cases been K. A. M. et unrelated cord blood transplantation for chronic granulomatous disease: a case report and review of the 2009; PubMed Scopus Google the first case report of HSCT of an patient using preimplantation female typing and HLA-typing J. de Velde H. De Rycke M. et bone marrow transplantation for X-linked chronic granulomatous disease by using preimplantation female typing and HLA 2008; PubMed Scopus Google we 2 patients who underwent transplantation from a matched healthy sibling by combined PGD and HLA typing. We to PGD female to the to the of unaffected embryos that be from affected when using in for female typing should be and with parents in cases suitable MSD is available and transplantation using an unrelated donor the of a sibling donor or is not of the of a suitable The methodology be in patients HSCT, such as patients with a genetic disease or children who require transplantation in the future of of an such as in first complete I. et the of genetic diagnosis with in 2009; PubMed Scopus Google In the parents of the affected be in Before parents should be informed the and of such as HSCT using an or an unrelated UCB should be informed the possibility of using BM or reduced-volume BM as in patients when insufficient UCB cells are A cell × is to engraftment in patients with diseases C. G. et cord blood the experience from Google Scholar]. in patient the UCB cells been to secure engraftment without the of BM we to combine UCB and BM to increase the cell of the limited and not experience with in patients with X-CGD patients K. A. M. et unrelated cord blood transplantation for chronic granulomatous disease: a case report and review of the 2009; PubMed Scopus Google Scholar]. BM from a donor, is an and should be only after UCB or transplantation In such cases, we transplantation the donor is the ages of this age on the a reduced BM volume not donor should to a of engraftment in the of insufficient UCB of stem cells et of parental stem cells to engraftment in recipients of unrelated donor umbilical cord of a clinical 2009; PubMed Scopus (194) Google or CD34+ cells G. C. et blood by of hematopoietic stem cells from a 2009; PubMed Scopus Google Scholar]. the available are insufficient to such a strategy as an to BM cells obtained from the same donor to increase the engraftment potential of the UCB transplant should be of the and of the PGD/HLA typing as well as the of its use. of the an for a suitable unrelated donor at the time of the for HSCT should be IntroductionX-linked chronic granulomatous disease (X-CGD) is an inherited defect in phagocyte nicotinamide adenine dinucleotide phosphate oxidase resulting from a deficiency of gp91phox component. At the molecular level, X-CGD is caused by mutations affecting the CYBB gene on chromosome Xp21.1 [1Heyworth P.G. Cross A.R. Curnutte J.T. Chronic granulomatous disease.Curr Opin Immunol. 2003; 15: 578-584Crossref PubMed Scopus (332) Google Scholar, 2Segal B.H. Leto T.L. Gallin J.I. et al.Genetic, biochemical, and clinical features of chronic granulomatous disease.Medicine (Baltimore). 2000; 79: 170-200Crossref PubMed Scopus (720) Google Scholar]. As a result of impaired phagocytic ability, affected persons suffer from severe recurrent infections, both bacterial and fungal, resulting in the formation of infiltrating granulomatous lesions. Despite prophylactic therapy, long-term survival remains poor; only 50% of patients are alive at the age of 30 years [3Van den Berg J.M. van Koppen E. Ǻhlin A. et al.Chronic granulomatous disease: The European experience.PLoS ONE. 2009; 4 (e5234:1–10)Crossref Scopus (497) Google Scholar, 4Winkelstein J.A. Marino M.C. Johnston Jr., R.B. et al.Chronic granulomatous disease. Report on a national registry of 368 patients.Medicine (Baltimore). 2000; 79: 155-169Crossref PubMed Scopus (1223) Google Scholar]. Allogeneic hematopoietic stem cell transplantation (HSCT) using an HLA-matched sibling donor (MSD) has achieved long-term cure of X-CGD in 80% to 90% of cases [5Seger R.A. Güngor T. Belohradsky B.H. et al.Treatment of chronic granulomatous disease with myeloablative conditioning and an unmodified hemopoietic allograft: a survey of the European experience, 1985-2000.Blood. 2002; 100: 4344-4350Crossref PubMed Scopus (226) Google Scholar, 6Seger R.A. Modern management of chronic granulomatous disease.Br J Haematol. 2008; 140: 255-266Crossref PubMed Scopus (194) Google Scholar]. A healthy MSD is not available in the vast majority of the cases, however. For such patients, a matched unrelated donor (MUD) is a suitable option, but at the cost of increased teatment-related morbidity and mortality (TRM) [5Seger R.A. Güngor T. Belohradsky B.H. et al.Treatment of chronic granulomatous disease with myeloablative conditioning and an unmodified hemopoietic allograft: a survey of the European experience, 1985-2000.Blood. 2002; 100: 4344-4350Crossref PubMed Scopus (226) Google Scholar, 7Del Giudice I. Iori A.P. Mengarelli A. et al.Allogeneic stem cell transplant from HLA-identical sibling for chronic granulomatous disease and review of the literature.Ann Hematol. 2003; 82: 189-192PubMed Google Scholar]. Preimplantation genetic diagnosis (PGD) for single-gene diseases was developed to help families desiring a new pregnancy to select unaffected embryos in vitro, thereby avoiding the need for pregnancy termination. Recently, this strategy has been combined with HLA typing with the aim of selecting suitable histocompatible embryos as potential stem cell donors for affected siblings [8Fiorentino F. Biricik A. Karadayi H. et al.Development and clinical application of a strategy for preimplantation genetic diagnosis of single gene disorders combined with HLA matching.Mol Hum Reprod. 2004; 10: 445-460Crossref PubMed Scopus (70) Google Scholar, 9Van de Velde H. Georgiou I. De Rycke M. et al.Novel universal approach for preimplantation genetic diagnosis of β-thalassemia in combination with HLA matching of embryos.Hum Reprod. 2004; 19: 700-708Crossref PubMed Scopus (92) Google Scholar, 10Kuliev A. Rechitsky S. Tur-Kaspa I. et al.Preimplantation genetics. Improving access to stem cell therapy.Ann N Y Acad Sci. 2005; 1054: 223-227Crossref PubMed Scopus (47) Google Scholar]. There is limited experience with this methodology and even less with its completed clinical application, however.